Broad spectrum inhibitors

ABSTRACT

The invention features a method of designing broad spectrum inhibitors using structural data, compositions having broad spectrum activity, and methods for treating disease using those compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims benefit of U.S. Provisional ApplicationNo. 0/344,788, filed Jan. 7, 2002, and No. 60/383,575, filed May 29,2002, each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the field of inhibitors andmethods for identifying or designing broad spectrum therapeutics for usein the treatment of infectious diseases and cancers, particularly wheredrug resistance is, or could reasonably predicted to be, an obstacle tosuccessful long term therapy.

[0003] The development of drug resistance is one of the most commoncauses of drug failure in the treatment of diseases involvingreplicating biological entities (i.e., cancer and infectious diseases).Drug resistance often results from a reduction in drug-binding affinityand can be quantified by the ratio of drug binding affinity (Kd) forvariant and wild type target proteins. Administration of a drugintroduces a selective pressure upon the replicating biological entity.The result is the emergence of drug resistant strains.

[0004] Drug resistance is a major obstacle to the successful treatmentof many cancers and infections, both bacterial and viral. For example,increased resistance of bacterial infections to antibiotic treatment hasbeen extensively documented and has now become a generally recognizedmedical problem, particularly with nosocomial infections. See, forexample, Jones et al., Diagn. Microbiol. Infect. Dis. 31:379 (1998);Murray, Adv. Intern. Med. 42:339 (1997); and Nakae, Microbiologia 13:273(1997).

[0005] Drug resistance has complicated the treatment for HIV as newmutant strains of HIV have emerged that are resistant to multiple,structurally diverse, experimental and chemotherapeutic antiretrovirals,including HIV protease inhibitors (PIs), nucleoside and non-nucleosideHIV reverse transcriptase inhibitors (NRTIs and NNRTIs), and HIV fusioninhibitors (FIs).

[0006] More than 60 million people have been infected by HIV in the lasttwo decades, and 20 million people have died from HIV/AIDS. While thedevelopment of highly active antiretrovirals to treat HIV/AIDS has ledto significant reductions in the mortality and morbidity of AIDS, therapid emergence and spread of drug-resistant mutant strains of HIV isrendering current drugs ineffective, and is the major cause of treatmentfailure. Recent estimates are that nearly 50% of drug-experiencedpatients in North America harbor HIV that is resistant to one or more ofthe 16 FDA-approved antiretroviral agents used in multi-drug ‘cocktails’(Ref. dont have this ref). Moreover, it has been estimated thatdrug-resistant HIV accounts for up to 12% of new infections (Little etal., N. Engl. J. Med., 347:385 (2002)).

[0007] Accordingly, drug resistant HIV strains represent distinctinfectious entities from a therapeutic viewpoint, and pose newchallenges for drug design as well as drug treatment of existinginfections. Substitutions have been documented in over 45 of the 99amino acids of the HIV protease monomer in response to proteaseinhibitor treatment (Mellors, et al., International Antiviral News, 3:8(1995); Eastman, et al., J. Virol., 72:5154 (1998); Kozal, et al., Nat.Med., 2:753 (1996)). The particular sequence and pattern of mutationsselected by PIs is believed to be somewhat drug-specific and oftenpatient-specific, but high level resistance is typified by multiplemutations in the protease gene which give rise to cross-resistance toall of the PIs.

[0008] In view of the foregoing problems, there exists a need forinhibitors against drug resistant and mdrHIV strains. Further, thereexists a need for inhibitors against drug resistant and multi-drugresistant HIV proteases (mdrPR). Further still, there exists a need forinhibitors of HIV that can prevent or slow the emergence of drugresistant and mdrHIV strains in infected individuals.

[0009] Inhibitors with the ability to inhibit mdrHIV strains, and toslow the emergence of drug resistant strains in wild type HIVinfections, are defined as “resistance-repellent” inhibitors.

[0010] There also exists a need for robust methods that can be used todesign “resistance-repellent” inhibitors.

[0011] More generally, there is a need for therapeutic regimens thatminimize the likelihood that resistance will occur in a diseaseinvolving a replicating biological entity. In one approach, drugs may bedesigned which have similar activity against both the wild type andmutant forms of their target. Such drugs minimize the probability of amutant population emerging by reducing the selective pressure introducedby the drug when used to treat wild type infections. Such drugs also canbe used to treat mutant infections and can be used for salvage therapy.

[0012] There is also an urgent need to develop potent, broad-spectrum,and mechanistically-novel antimicrobials suitable for tackling thegrowing problem of antibiotic-resistant bacteria strains, and fortreating and/or preventing outbreaks of infectious diseases, includingdiseases caused by bioterrorism agents like anthrax, plague, cholera,gastroenteritis, multidrug-resistant tuberculosis (MDR TB). The recentanthrax attack of 2001 underscored the reality of large-scale aerosolbioweapons attack by terrorist groups. It also revealed that there is anurgent and pressing need to discover and develop novel and potentantimicrobials that can be used therapeutically and prophylactically forbiodefense against new bioattacks. The NIH and CDC have identified anumber of High Priority pathogens based on their likelihood of causingwidespread contagious disease and/or death to the general population.Research on methods of protection against potential agents ofbioterrorism has been a priority for several years at the NIH. A recentanalysis suggested the existence of ongoing offensive biological weaponsprograms in at least 13 countries (Inglesby, T. V., et al., JAMA,287:2236, (2002)).

[0013] The widespread use of antibiotics in human medical as well as inagricultural applications has promoted the emergence and spread of drugresistant bacteria that are no longer sensitive to existing drugs. Theease with which drug resistant microorganisms can be selected in asimple laboratory setting is a further concern when contemplatingpharmaceutical-based strategies for biodefense. There is an urgent needto discover and develop novel therapeutic agents to combat pathogensthat are likely to be used in a bioterrorist scenario.

[0014] A list of selected agents rated by likelihood to cause thegreatest harm in a bioterrorist attack has been compiled by the CDC andNIAID (Lane, H. C., et al., Nat Med., 7:1271 (2001)). B. anthracis; thebacterium that causes anthrax, is one of the most serious of the group Apathogens. Dissemination of B. anthracis spores via the US PostalService in 2001 established the feasibility of large-scale aerosolbioweapons attack. It has been estimated that between 130,000 and 3million deaths would follow the release of 100 kg of B. anthracis, alethality matching that of a hydrogen bomb (Inglesby, T. V., et al.,JAMA, 287:2236, (2002)). Penicillin, doxycycline and ciprofloxacin arecurrently approved by the FDA for the treatment of inhalation anthraxinfections. However, it was advised that antibiotic resistance topenicillin- and tetracycline-class antibiotics should be assumedfollowing a terrorist attack (Inglesby, T. V., et al., JAMA, 281:1735-45(1999)). Moreover, in vitro selection of a B. anthracis strain that isresistant to ofloxacin (a fluoroquinilone closely related tociprofloxacin) has been reported (Choe, C. H., et al., Antimicrob.Agents. Chemother., 44:1766 (2000)). Following the anthrax attacks of2001, the CDC advocated the use of a combination of 2-3 antibiotics. Asa post-exposure prophylaxis, 60 days of treatment with ciprofloxacin iscurrently recommended. Strict compliance to this drug regimen iscomplicated by moderate to severe gastrointestinal tract intolerance.

[0015] Another group A pathogen, Y. pestis, is the causative agent ofplague. If 50 kg of Y. pestis were released as an aerosol over a city of5 million, pneumonic plague would afflict an estimated 150,000individuals and result in 36,000 deaths (Inglesby, T. V., et al., JAMA,283: 2281, (2000)). Streptomycin, tetracycline and doxycycline are theFDA-approved treatment for plague. Wide spread use of these antibioticsin the US raises concerns about possible resistance. A US-licensed,formaldehyde-killed whole bacilli vaccine was discontinued by itsmanufacturers in 1999 and is no longer available.

[0016]C. jejuni and V. cholerae are category B pathogens which canpresent a significant threat to the safety of food and water supplies.C. jejuni infections are one of the most commonly identified causes ofacute bacterial gastroenteritis worldwide and area frequent cause ofTraveler's diarrhea (Allos, B. M., Clin Infect Dis, 32:1201 (2001)).Currently, the CDC estimates that 2.4 million cases of Campylobacterinfection occur in the United States each year, affecting almost 1% ofthe entire population. In the past few years, a rapidly increasingproportion of Campylobacter strains all over the world have been foundto be fluoroquinolone-resistant. High rates of resistance maketetracycline, amoxicillin, ampicillin, metronidazole, and cephalasporinspoor choices for the treatment of C. jejuni infections. AllCampylobacter species are inherently resistant to vancomycin, rifampin,and trimethoprim. V. cholerae, a causative agent of cholera, isresponsible for 120,000 deaths annually (Faruque, S. M., et al.,Microbiol Mol Biol Rev, 62:1301 (1998)) and is characterized by arapidly changing pattern of antibiotic resistance.

[0017] TB is one of the most common and devastating infectious diseasesknown to man. An estimated one third of the global population isinfected with Mycobacteria tuberculosis. Eight million people develop anactive infection and 2 million victims die yearly (Dye, C., et al.,JAMA, 282:677 (1999.)). Currently, a combination of four drugs isrecommended for TB treatment: isoniazid, rifampicin, pyrazinamide andethambutole. The treatment course lasts 6 months. Such a multidrugcombination together with the lengthy duration of treatment is prone toside-effects and adherence problems, which in turn can often lead to thedevelopment of drug resistance. The current drugs used to treat TBinfections were introduced into clinical practice more than 30 yearsago, in the absence of any knowledge of molecular mechanism. There is anurgent need to identify novel, effective, non-toxic and specific drugsthat can shorten the duration of treatment, reduce side-effects, combatlatent infection and reduce the spread of MDR TB strains. In addition,it is important to recognize the need for mechanistically novel drugs,i.e., antimicrobial agents that target biochemical pathways distinctfrom those of existing TB drugs, in order to be effective against MDR TBstrains.

[0018] In summary, there is a clear need for the discovery of novel,non-toxic, broad spectrum antibiotics that can be used to (1) treatdrug-resistant bacterial infections, and (2) protect civilians andmilitary personnel in case of bioterrorist attacks. In one approach,drugs may be designed which have similar activity against both the wildtype and variant forms of their target. Such drugs should minimize theprobability of the emergence of mutant populations by reducing theselective pressure introduced by the drug when used to treat wild typeinfections. Such drugs also can be used to treat mutant infections andcan be used for salvage therapy. In another approach, drugs may bedesigned which have similar activity against various isotypes of ahomologous target. Such drugs can be used to treat multiple species ofpathogenic microorganisms since they will be active against the targetof each species. In a third approach, drugs can be designed that combinethe properties and the uses of both of the above approaches.

[0019] There also exists a need for robust methods that can be used todesign such broad spectrum antibiotics.

SUMMARY OF THE INVENTION

[0020] In a first aspect the invention features a method for thestructure-based design of a drug that can act as an inhibitor of atleast two different biological entities, the method comprising the stepsof: (a) providing at least one structure of a wild type target proteinor an inhibitor-wild type target protein complex; (b) providing at leastone structure of a variant target protein or an inhibitor-variant targetprotein complex; (c) comparing at least one structure from step (a) withat least one structure from step (b) to determine whether there exists acommon three-dimensionally conserved substructure comprising the atomiccoordinates of the structurally conserved atoms of the inhibitors andstructurally conserved atoms of the target proteins; and (d) if aconserved substructure exists, using the atomic coordinates of theconserved substructure to select a compound having atoms matching thoseof the structurally conserved atoms of the inhibitors, wherein theselection of the compound is performed using computer modeling.

[0021] The invention also features a method for the structure-based drugdesign of a broad spectrum compound, the method comprising the steps of:(a) providing at least one structure of a wild type target protein or aninhibitor-wild type target protein complex; (b) providing at least onestructure of a variant target protein or an inhibitor-variant targetprotein complex; (c) comparing at least one structure from step (a) withat least one structure from step (b) to determine whether there exists acommon three-dimensionally conserved substructure comprising the atomiccoordinates of the structurally conserved atoms of the target proteinsor a common three-dimensionally conserved substructure comprising theatomic coordinates of the structurally conserved atoms of the inhibitorsand structurally conserved atoms of the target proteins; and (d) if aconserved substructure exists, using the atomic coordinates of theconserved substructure to select a compound having atoms matching thoseof the structurally conserved atoms of the inhibitors or to design acompound that binds to the target protein, wherein the selection of thecompound is performed using computer modeling.

[0022] Desirably, the above method further comprises the steps of: (e)comparing at least one structure from step (a) with at least onestructure from step (b) to determine whether there exists athree-dimensionally non-conserved substructure comprising the atomiccoordinates of the structurally non-conserved atoms of the inhibitorsand structurally non-conserved atoms of the target proteins; and (f) ifa non-conserved substructure exists, using the atomic coordinates of thenon-conserved substructure to reject a compound having atoms matchingthose of the structurally non-conserved atoms of the inhibitors, whereinthe rejection of the compound is performed in conjunction with computermodeling.

[0023] In any of the above methods, at least two, four, six, or eightstructures from step b can be used in step c. The methods can be appliedusing several structures, including at least two, four, six, or eightvariant forms of the target protein.

[0024] The inhibitors used in the inhibitor-wild type target proteincomplex and the inhibitor-variant target protein complex can be the sameor different. The inhibitors can be selected from competitive ornoncompetitive inhibitors. Furthermore, the inhibitors can be selectedfrom reversible, or irreversible inhibitors.

[0025] In any of the above methods, the variant target protein can be ahomologous protein or a mutant protein.

[0026] In any of the above methods, the structures can be selected fromcrystal structures, NMR structures, computer models, any acceptableexperimental, theoretical or computational method of deriving athree-dimensional representation of a structure, or a combinationthereof.

[0027] Target proteins for use in the present invention include anytherapeutically relevant protein. The target protein can be a viral,bacterial, protozoan, or fungal protein. In some instances, the targetprotein is one that is expressed in a neoplasm.

[0028] Preferably, the target protein can be an enzyme, a receptor, astructural protein, a component of a macromolecular complex, a componentof a metabolic pathway, or an assembly of biological molecules.Desirably, the target protein is necessary for the survival of thereplicating biological entity. For example, the target protein can be anenzyme selected from the group consisting of reverse transcriptases,proteases, DNA and RNA polymerases, methylases, oxidases, esterases,acyl transferases, helicases, topoisomerases, and kinases. The targetprotein can be a component of a metabolic pathway, such as the shikimatepathway. Desirable target proteins include HIV protease or3-dehydroquinate dehydratase, among others.

[0029] Where the target protein is HIV protease, suitable inhibitors foruse in the methods of the invention include those selected from thegroup consisting of indinavir, nelfinavir, ritonavir, saquinavir,amprenavir, lopinavir, and UIC-94003.

[0030] A broad spectrum protease inhibitor can be designed using thesusbstructure of structurally conserved atoms described by the atomiccoordinates in Table 8, which includes the structurally conserved atomsof the inhibitor and structurally conserved atoms of the protease. Abroad spectrum protease inhibitor can also be designed using thestructurally conserved atoms of the inhibitor alone. These are describedby the atomic coordinates in Table 8.

[0031] A broad spectrum 3-dehydroquinate dehydratase inhibitor can bedesigned using the susbstructure of structurally conserved atomsdescribed by the atomic coordinates in Table 12, which includes thestructurally conserved atoms of the 3-dehydroquinate dehydratase. Abroad spectrum 3-dehydroquinate dehydratase inhibitor can also bedesigned using the structurally conserved atoms of the inhibitor alone.These are described by the atomic coordinates in Table 12.

[0032] The invention also features compounds having a chemical structureselected using any of the methods above. Such compounds are broadspectrum inhibitors and have broad spectrum activity against replicatingbiological entities expressing a particular target protein. Thus, if thetarget protein is expressed by a microbe or a neoplasm, the compoundwill have broad spectrum activity against the microbe or neoplasm,respectively.

[0033] The invention features a compound having broad spectrum activityagainst HIV protease wherein the compound has a chemical structureselected using the methods above, including those methods utilizing theatomic coordinates of Table 8.

[0034] The invention features a compound having broad spectrum activityagainst 3-dehydroquinate dehydratase wherein the compound has a chemicalstructure selected using the methods above, including those methodsutilizing the atomic coordinates of Table 12.

[0035] The compounds of the invention exclude bis-THF compounds (e.g.,analogs of compounds 1 and 3) as described in J. Med. Chem. 39:3278-3290(1996) (compounds 49-52 and 58-60), Bioorg. Med. Chem. Lett. 8:979-982(1998), WO99/65870, U.S. Pat. No. 6,319,946, WO02/08657, WO02/092595,WO99/67417, EP00/9917, and WO00/76961; and also exclude fused ring THFstructures as described in Bioorg. Med. Chem. Lett. 8:687-690 (1998) andU.S. Pat. No. 5,990,155.

[0036] For any of the broad spectrum inhibitors of the invention, broadspectrum activity can be measured by the ratio of the inhibitoryconcentrations of the broad spectrum inhibitor for the variant and wildtype biological entities (IC_(50, variant)/IC_(50, wild type)).Desirably, the IC_(50, variant)/IC_(50, wild type) ratio for a broadspectrum inhibitor is less than 100, 80, 60, 40, 30, 20, 10, 8, 6, or,most desirably, less than 3.

[0037] A broad spectrum inhibitor can be active against severaldifferent mutant biological entities. Desirably, the inhibitor will havebroad spectrum activity against at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 mutant biological entities.

[0038] A broad spectrum inhibitor can also be active against differentorganisms or neoplastic cell types expressing homologous target proteinsthat possess sufficient structural similarity. Desirably, the inhibitorwill have broad spectrum activity against at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, or 20 different organisms or neoplastic celltypes expressing homologous target proteins.

[0039] The invention also features a pharmaceutical composition thatincludes a broad spectrum inhibitor described herein in anypharmaceutically acceptable form, including isomers such asdiastereomers and enantiomers, salts, solvates, and polymorphs thereof.The composition can include an inhibitor of the invention along with apharmaceutically acceptable carrier or diluent.

[0040] The invention also features methods of treating disease in apatient in need thereof, which includes the administration of apharmaceutical composition of the invention to the patient in an amountsufficient to treat the disease. The pharmaceutical composition includesany broad spectrum inhibitor described herein. Such broad spectruminhibitors have broad spectrum activity against replicating biologicalentities expressing a particular target protein. Thus, if the targetprotein is expressed by a microbe or a neoplasm, the disease to betreated will be a microbial infection or neoplasm, respectively.

[0041] The invention features a method of treating an HIV infection in apatient in need thereof, the method including the step of administeringto the patient a pharmaceutical composition including a broad spectrumprotease inhibitor described herein in amounts effective to treat theHIV infection.

[0042] The invention features a method of treating a bacterial infectionin a patient in need thereof, the method including the step ofadministering to the patient a pharmaceutical composition including abroad spectrum 3-dehydroquinate dehydratase inhibitor described hereinin amounts effective to treat the bacterial infection. The bacterialinfection to be treated using the above method can be caused by abacterium selected from the group consisting of C. jejuni, V. cholerae,Y. pestis, B. anthracis, P. putidas, and M. tuberculosis. Furthermore,this method can be used to treat infections by any microbe the utilizes3-dehydroquinate dehydratase.

[0043] The invention also features the use of a pharmaceuticalcomposition described herein in the manufacture of a medicament for thetreatment of a disease. The pharmaceutical composition includes anybroad spectrum inhibitor described herein. Such broad spectruminhibitors and have broad spectrum activity against replicatingbiological entities expressing a particular target protein. Thus, if thetarget protein is HIV protease or 3-dehydroquinate dehydratase, thedisease to be treated will be an HIV infection or bacterial infection,respectively.

[0044] The term “replicating biological entity” includes, for example,bacteria, fungi, yeasts, viruses, protozoa, prions and neoplasms

[0045] Neoplasms include, for example, carcinomas of the bladder,breast, colon, kidney, liver, lung, head and neck, gall-bladder, ovary,pancreas, stomach, cervix, thyroid, prostate, or skin; a hematopoietictumor of lymphoid lineage; a hematopoietic tumor of myeloid lineage; atumor of mesenchymal origin; a tumor of the central or peripheralnervous system; melanoma; seminoma; teratocarcinoma; osteosarcoma;thyroid follicular cancer; and Kaposi's sarcoma. Hematopoietic tumors oflymphoid lineage can be leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett'slymphoma.

[0046] By “wild type target protein” is meant a protein obtained from areplicating biological entity that has not been subjected to drugselection pressure, and could include polymorphisms or isoforms thereof.A replicating biological entity that expresses wild type target proteinis referred to herein as a wild type biological entity.

[0047] By “variant target protein” is meant a mutant target protein or ahomologous target protein. A replicating biological entity thatexpresses variant target protein is referred to herein as a variantbiological entity.

[0048] By “mutant target protein” is meant a target protein thatcontains one or more amino acid substitutions with respect to the wildtype target protein, including proteins from the same organism that haveevolved under drug selection pressure. In general, mutant targetproteins will have one or more amino acid substitutions and should bereadily identified as related to the cognate wild type protein usingstandard sequence comparison methods. A replicating biological entitythat expresses mutant target protein is referred to herein as a mutantbiological entity.

[0049] By “homologous target protein” is meant a variant target proteinthat is expressed in a different species or neoplastic cell type thanthe wild type target protein, but has the same, or similar, function.

[0050] By “structurally conserved target substructure”, and by“structurally” or “three-dimensionally conserved substructure” asapplied to target proteins, is meant the regions of the target proteinstructure which are not significantly affected by amino acid mutationsor substitutions. Such regions can be defined using standard methods ofcomparative analysis of three-dimensional structures of proteins, suchas superposition analysis, for example. In the case of HIV protease,these regions were identified using a pair wise superposition analysisof wild type and mutant protease structures complexed with inhibitors.The superposition of structures can be performed using the iterativeprocedure described herein. In the case of DHQase, these regions wereidentified using a pair wise superposition analysis of wild type andhomologous DHQase structures from different bacterial species with andwithout inhibitors. It is apparent that the overall compositions ofstructurally conserved target substructures will likely differ fordifferent, non-homologous target proteins, especially when the frequencyof amino acid substitutions in high. However, a quantitative definitioncan be derived from the superposition analysis, which provides both theidentities and the positions of the atoms that comprise thesesubstructures. The regions that comprise structurally conserved targetsubstructures contain atoms whose superimposed pairs havethree-dimensional atomic coordinates that match to within a distance of1 Å, 0.6 Å, 0.4 Å, or 0.2 Å.

[0051] By “broad spectrum inhibitor” is meant a compound having broadspectrum activity, i.e., an inhibitor that is active against twodifferent-biological entities, e.g., both a wild type biological entityand one or more variants of that biological entity. Thus, broad spectrumactivity can be described by the inhibitor's action against a particulartarget protein (e.g., broad spectrum activity against protease) or aparticular target organism (e.g., broad spectrum activity against HIV).Broad spectrum inhibitors will have medically insignificant interactionswith non-conserved regions. Broad spectrum inhibitors can be useful forthe treatment and/or prevention of infectious diseases caused bymultiple infectious agents, as well as for decreasing the development ofdrug-resistance by these organisms.

[0052] As used herein, the term “treating” refers to administering apharmaceutical composition for prophylactic and/or therapeutic purposes.To “prevent disease” refers to prophylactic treatment of a patient whois not yet ill, but who is susceptible to, or otherwise at risk of, aparticular disease. To “treat disease” or use for “therapeutictreatment” refers to administering treatment to a patient alreadysuffering from a disease to ameliorate the disease and improve thepatient's condition. Thus, in the claims and embodiments, treating isthe administration to a patient either for therapeutic or prophylacticpurposes.

[0053] The term “microbial infection” refers to the invasion of the hostpatient by pathogenic microbes (e.g., bacteria, fungi, yeasts, viruses,protozoa). This includes the excessive growth of microbes that arenormally present in or on the body of a patient. More generally, amicrobial infection can be any situation in which the presence of amicrobial population(s) is damaging to a host patient. Thus, a patientis “suffering” from a microbial infection when excessive numbers of amicrobial population are present in or on a patient's body, or when-thepresence of a microbial population(s) is damaging the cells or othertissue of a patient.

[0054] The term “microbes” includes, for example, bacteria, fungi,yeasts, viruses and protozoa. The term “administration” or“administering” refers to a method of giving a dosage of apharmaceutical composition to a patient, where the method is, e.g.,topical, oral, intravenous, intraperitoneal, or intramuscular. Thepreferred method of administration can vary depending on variousfactors, e.g., the components of the pharmaceutical composition, site ofthe potential or actual disease and severity of disease.

[0055] The term “patient” includes humans, cattle, pigs, sheep, horses,dogs, and cats, and also includes other vertebrate, most preferably,mammalian species.

[0056] Where “atomic coordinates” are provided, or otherwise referredto, these coordinates define a three dimensional structure. That such astructure may be defined by more than one different coordinate system,e.g., by translation or rotation of the coordinates, does not change therelative positions of the atoms in the structure. Accordingly, anyreference to atomic coordinates herein is intended to include anyequivalent three dimensional structure defined by the coordinates.

[0057] By “computer modeling” is meant the use of a computer tovisualize or compute a compound, a portion of a compound, a targetprotein, a portion of a target protein, a complex between a compound anda target protein, or a portion of a complex between a compound and atarget protein.

[0058] Other features and advantages of the invention will be apparentfrom the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1 is a table depicting the structures of compounds 1-7, gt33,and qxa.

[0060]FIG. 2 illustrates the amino acid alignment of type II DHQases.Fully conserved residues are framed. Catalytically important amino acidsare marked by stars. Arrows denote amino acids that make hydrogen bondsand ionic interactions in the structure of M. tuberculosis DHQasecomplexed with the inhibitor, 3-dehydroquinic acid oxime.

[0061]FIG. 3 illustrates the key interactions of the substrate-basedinhibitor, DHQO, with the active site residues for the Type II DHQasefrom M. tuberculosis.

DETAILED DESCRIPTION

[0062] We have discovered that the comparative analysis of thestructures of complexes of inhibitors bound to wild type and variantforms of a target protein can be used to design compounds that are broadspectrum inhibitors.

[0063] The methods of the invention entail the design of compoundshaving a particular structure. The methods rely upon the use ofstructural information to arrive at these compounds. The structural datadefine a three dimensional array-of the important contact atoms in aninhibitor that bind to the target protein in a fashion that results inbroad spectrum activity against biological entities expressing variantsof the target protein.

[0064] Inhibitor-Target Protein Structures

[0065] Atomic structural coordinates can be selected from crystalstructures, NMR structures, computer models, any acceptableexperimental, theoretical or computational method of deriving athree-dimensional representation of a structure, or a combinationthereof. Atomic coordinates for use in the methods of the invention canbe obtained from publicly available sources, e.g. from the Protein DataBank, or obtained using known experimental or computational methods.

[0066] Atomic structural coordinates for use in the methods of theinvention include crystal structures of HIV protease/inhibitor complexesderived from wild type and drug-resistant mutant proteases, and ofDHQase and DHQase inhibitor complexes derived from two or more bacterialspecies, among others. In examples 1-3, the methods of the invention areapplied using the coordinates of wild type HIV protease complexed withamprenavir, wild type HIV protease complexed with UIC-94003, andV82F/184V mutant HIV protease complexed with UIC-94003. In example 4,the methods of the invention are applied using the coordinates of wildtype DHQase from M. tuberculosis and from Pseudomonas putidas. a complexbetween a compound and a target protein. The coordinates of otherrepresentative structures of HIV protease and DHQase should be usefulfor performing the methods of the present invention.

[0067] Conserved Substructures

[0068] Conserved substructures can be identified for target proteins,for target protein-inhibitor complexes, and/or for inhibitors, dependingon the nature of the structures that are used in the comparativesuperposition analysis. In one approach, at least one structure of awild type target protein is compared to at least one structure of amutant or homologous target protein to determine whether a commonthree-dimensionally conserved substructure is present among the wildtype protein and the mutant or homologous proteins, respectively. Inanother approach, at least one structure of an inhibitor-wild typetarget protein complex and at least one structure of an inhibitor-mutanttarget protein complex are compared to determine whether a commonthree-dimensionally conserved substructure is present among the mutantand wild type complexes. In a third approach, at least one structure ofan inhibitor-wild type target protein complex and at least one structureof a mutant or homologous target protein without inhibitor are comparedto determine whether a common three-dimensionally conserved substructureis present among the respective mutant or homologous protein and thewild type complexes. Variations of the approached described above canalso be used. In each case, such a comparison can be made by means of(a) an overall superposition of the atoms of the protein structures;and, where feasible, (b) a study of the distances from atoms of theinhibitors to atoms of the protein. This analysis requiresthree-dimensional atomic coordinates of the protein structures and ofthe bound inhibitor.

[0069] The superposition of the protein structures can be performed in atwo step process: 1) the distance between all pairs of corresponding Cáatoms (Cá atom of residue number 1 in one protein to Cá atom of residuenumber 1 in the second protein; Cá atom of residue number 2 in oneprotein to Cá atom of residue number 2 in the second protein; and so on)of the polypeptide chains is minimized by means of a least-squarealgorithm; 2) the superposition is refined by minimizing, in aniterative process, the distances between corresponding Cá atoms that arecloser than a given distance (0.25 A for example), thus eliminatingregions of the structures having large conformational differences tocompute the superposition parameters. Furthermore, where a partialstructure is provided (e.g., from NMR data) the available coordinatesare superimposed.

[0070] The conserved substructure identifies the relevant portion of thetarget protein that is the active site, or binding region, defined bythat part of the target protein interacting with inhibitor. Importantinteractions between the target protein and inhibitor are identified bymapping the contacts between the two. Structurally conserved regions ofthe target protein not near the binding site are generally not relevantto the design of the broad spectrum inhibitor. Accordingly, theselection of the meaningful substructure is identified using the abovementioned contacts.

[0071] Design of a Broad Spectrum Inhibitor

[0072] The coordinates of the conserved inhibitor substructure are usedto design an inhibitor having atoms matching those of thethree-dimensionally structurally conserved atoms of the inhibitors. Theresult is an inhibitor for which IC_(50, variant) and IC50, wild typeare similar, minimizing the selective pressure introduced by the drug.

[0073] The methods of the invention can employ computer-based methodsfor designing broad spectrum inhibitors. These computer-based methodsfall into two broad classes: database methods and de novo designmethods. In database methods the compound of interest is compared to allcompounds present in a database of chemical structures and compoundswhose structure is in some way similar to the compound of interest areidentified. The structures in the database are based on eitherexperimental data, generated by NMR or x-ray crystallography, or modeledthree-dimensional structures based on two-dimensional (i.e., sequence)data. In de novo design methods, models of compounds whose structure isin some way similar to the compound of interest are generated by acomputer program using information derived from known structures, e.g.,data generated by x-ray crystallography and/or theoretical rules. Suchdesign methods can build a compound having a desired structure in eitheran atom-by-atom manner or by assembling stored small molecularfragments.

[0074] The success of both database and de novo methods in identifyingcompounds having the desired activity depends on the identification ofthe functionally relevant portion of the compound of interest. Thefunctionally relevant portion of the compound, the pharmacophore, isdefined by the structurally conserved substructure. A pharmacophore thenis an arrangement of structural features and functional groups importantfor obtaining an inhibitor having broad spectrum activity.

[0075] Not all identified compounds having the desired pharmacophorewill act as broad spectrum inhibitors. The actual activity can befinally determined only by measuring the activity of the compound inrelevant biological assays. However, the methods of the invention areextremely valuable because they can be used to greatly reduce the numberof compounds which must be tested to identify those likely to exhibitbroad spectrum activity.

[0076] Programs suitable for generating predicted three-dimensionalstructures from two-dimensional data include: Concord (TriposAssociated, St. Louis, Mo.), 3-D Builder (Chemical Design Ltd., Oxford,U.K.), Catalyst (Bio-CAD Corp., Mountain View, Calif.), and Daylight(Abbott Laboratories, Abbott Park, Ill.).

[0077] Programs suitable for searching three-dimensional databases toidentify molecules bearing a desired pharmacophore include: MACCS-3D andISIS/3D (Molecular Design Ltd., San Leandro, Calif.), ChemDBS-3D(Chemical Design Ltd., Oxford, U.K.), and Sybyl/3DB Unity (TriposAssociates, St. Louis, Mo.).

[0078] Programs suitable for pharmacophore selection and design include:DISCO (Abbott Laboratories, Abbott Park, Ill.), Catalyst (Bio-CAD Corp.,Mountain View, Calif.), and ChemDBS-3D (Chemical Design Ltd., Oxford,U.K.).

[0079] Databases of chemical structures are available from CambridgeCrystallographic Data Centre (Cambridge, U.K.) and Chemical AbstractsService (Columbus, Ohio).

[0080] De novo design programs include Ludi (Biosym Technologies Inc.,San Diego, Calif.) and Aladdin (Daylight Chemical Information Systems,Irvine Calif.).

[0081] One skilled in the art may use one of several methods to screenchemical entities for their ability to match the conserved substructure.This process may begin by visual inspection of, for example, the activesite on the computer screen based on the atomic coordinates for thetarget protein. Docking may be accomplished using software such asQuanta and Sybyl, followed by energy minimization and molecular dynamicswith standard molecular mechanics forcefields, such as CHARMM and AMBER.

[0082] Specialized computer programs may also assist in the process ofselecting chemical entities. These include:

[0083] 1. GRID (Goodford, P. J., “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules,” J. Med. Chem., 28:849 (1985)). GRID isavailable from Oxford University, Oxford, UK.

[0084] 2. MCSS (Miranker, A. and M. Karplus, “Functionality Maps ofBinding Sites: A Multiple Copy Simultaneous Search Method.” Proteins:Structure, Function, and Genetics, 11:29 (1991)). MCSS is available fromMolecular Simulations, Burlington, Mass.

[0085] 3. AUTODOCK (Goodsell, D. S. and A. J. Olsen, “Automated Dockingof Substrates to Proteins by Simulated Annealing,” Proteins: Structure,Function, and Genetics, 8:195 (1990)). AUTODOCK is available fromScripps Research Institute, La Jolla, Calif.

[0086] 4. DOCK (Kuntz, L. D. et al., “A Geometric Approach toMacromolecule-Ligand Interactions,” J. Mol. Biol., 161:269 (1982)). DOCKis available from University of California, San Francisco, Calif.

[0087] Once the conserved substructure for the inhibitor has beenidentified, the conserved atoms of the inhibitor can be selected forassembly into a single inhibitor. Assembly may be proceed by visualinspection of the relationship of the fragments to each other on thethree-dimensional image displayed on a computer screen in relation tothe structure coordinates of the target protein. This may be followed bymanual model building using software such as Quanta or Sybyl.

[0088] Useful programs to aid one of skill in the art in assembly of theindividual chemical entities or fragments include:

[0089] 1. CAVEAT (Bartlett, P. A. et al, “CAVEAT: A Program toFacilitate the Structure-Derived Design of Biologically ActiveMolecules”. In “Molecular Recognition in Chemical and BiologicalProblems,” Special Pub., Royal Chem. Soc., 78:182 (1989)). CAVEAT isavailable from the University of Calif., Berkeley, Calif.

[0090] 2. 3D Database systems such as MACCS-3D (MDL Information Systems,San Leandro, Calif.). This area is reviewed in Martin, Y. C., “3DDatabase Searching in Drug Design,” J. Med. Chem., 35:2145 (1992)).

[0091] 3. HOOK (available from Molecular Simulations, Burlington,Mass.).

[0092] Other molecular modeling techniques may also be employed inaccordance with this invention. See, e.g., Cohen, N. C. et al.,“Molecular Modeling Software and Methods for Medicinal Chemistry,” J.Med. Chem., 33:883 (1990). See also, Navia, M. A. and M. A. Murcko, “TheUse of Structural Information in Drug Design,” Current Opinions inStructural Biology, 2:202 (1992).

[0093] Once a broad spectrum inhibitor has been optimally designed, asdescribed above, substitutions may then be made in some of its atoms orside groups in order to improve or modify its binding properties.Generally, initial substitutions are conservative, i.e., the replacementgroup will have approximately the same size, shape, hydrophobicity andcharge as the original group. It should, of course, be understood thatcomponents known in the art to alter conformation should be avoided.

[0094] In general, inhibitors designed using the methods of theinvention can be tested for broad spectrum activity using any of the toin vitro and/or in vivo methods described below, among others.

[0095] Broad Spectrum Inhibitors

[0096] Broad spectrum inhibitors match the pharmacophore defined by thestructurally conserved substructure. The pharmacophore is thearrangement of structural features and functional groups important forobtaining an inhibitor having broad spectrum activity. Thispharmacophore is derived using structural data for known inhibitorscomplexed to a target protein. Accordingly, broad spectrum inhibitorswill often be structurally related to known compounds lacking broadspectrum activity, but useful in the design of broad spectrum inhibitorsusing the methods disclosed herein. These known inhibitors serve as leadcompounds for both the design and synthesis of a broad spectruminhibitor. Using the synthetic methods for making the lead compounds andstandard synthetic methods as described by, for example, J. March,Advanced Organic Chemistry: Reactions, Mechanisms and Structure,” JohnWiley & Sons, Inc., 1992; T. W. Green and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis” (2^(nd) Ed.), John Wiley & Sons, 1991; andP. J. Kocienski, “Protecting Groups,” Georg Thieme Verlag, 1994, one cansynthesize the broad spectrum inhibitors described herein.

[0097] Typically the lead compounds bear varied functional groups whichare present in the pharmacophore, including hydrogen-bond donors,hydrogen-bond acceptors, ionic moieties, polar moieties, hydrophobicmoieties, aromatic centers, and electron-donors and acceptors. These arelinked by a structural scaffold which imparts the appropriate a threedimension arrangement of the functional groups.

[0098] Numerous modifications of the lead compound can be made usingtechniques known in the art. These include changing a functional groupby replacing it with another moiety of the same group. For example, onehydrogen-bond donor may be substituted by another. A good hydrogen bonddonor has an H atom bonded to a very electronegative atom (e.g., O—H orN—H). Examples of hydrogen-bond donors include alcohols, carboxylicacids, oximes, and amides, among others. Similarly, one hydrogen-bondacceptor may be substituted by another. A good hydrogen bond acceptorhas an electronegative element with lone pairs (e.g., O, N, or F).Examples of hydrogen bond acceptors include water, halogen atoms,alcohols, amines, carbonyls, ethers, and amides, among others. It mayalso be desirable to alter the distance between functional groups in alead compound. This is achieved by employing synthetic methods analogousto those used to prepare the lead compound, but replacing the scaffoldwith a structurally related scaffold that provides the desired distance(e.g., a scaffold that incorporates more or fewer atoms linking therelevant functional groups). In some instances it may also be desirableto alter the stereochemistry in a lead compound. This can beaccomplished by employing racemic starting materials, or by employingreaction conditions that result in racemization of the relevant chiralcenter, followed by separation of the enantiomeric or diastereomericmixture.

[0099] Assays

[0100] Inhibitors designed using the methods disclosed herein may befurther assayed, using standard in vitro models or animal models, toevaluate therapeutic activity and toxicity. These assays are describedin the literature and are familiar to those skilled in the art. Theseinclude but are not limited to assays for monitoring or measuringefficacy against HIV, bacteria, and neoplasms.

[0101] One skilled in the art will be familiar with methods of measuringthe IC₅₀'s of a broad spectrum inhibitor described herein. The IC₅₀value is determined by plotting percent activity versus inhibitorconcentration in the assay and identifying the concentration at which50% of the activity (e.g., growth, enzymatic activity, proteinproduction, etc.) remains. Inhibitors can be tested for antimicrobialactivity against a panel of organisms according to standard proceduresdescribed by the National Committee for Clinical Laboratory Standards(NCCLS document 7-A3, Vol. 13, No. 25, 1993/NCCLS document M27-P, Vol.12, No. 25, 1992). Inhibitors can be dissolved (0.1 μg/ml-500 μg/ml) inmicrobial growth media, diluted, and added to wells of a microtiterplate containing bacteria or fungal cells in a final volume of anappropriate media (Mueller-Hinton Broth; Haemophilus Test Media;Mueller-Hinton Broth+5% Sheep Blood; or RPMI 1690). Typically, theplates are incubated overnight at an appropriate temperature (30° C. to37° C.) and optical densities (measure of cell growth) are measuredusing a commercial plate reader.

[0102] IC₅₀ 's for broad spectrum protease inhibitors can be measuredagainst wild type HIV and clinically isolated mutant HIV isolates,utilizing the PHA-PBMC exposed to HIV-1 (50 TCID₅₀ dose/X0⁶ PBMC) astarget cells and using the inhibition of p24 Gag protein production asan endpoint. The amounts of p24 antigen produced by the cells can bedetermined on day 7 in culture using a commercially availableradioimmunoassay kit. Drug concentrations resulting in 50% inhibition(IC₅₀'s) of p24 antigen production can be determined by comparison withthe p24 production level in drug-free control cell cultures.

[0103] Therapy

[0104] The invention features a method of identifying a compound havingbroad spectrum activity. Broad spectrum inhibitors of the presentinvention may be administered by any appropriate route for treatment orprevention of a disease or condition associated with a bacterialinfection, viral infection, or neoplastic disorder, among others. Thesemay be administered to humans, domestic pets, livestock, or otheranimals with a pharmaceutically acceptable diluent, carrier, orexcipient, in unit dosage form. Administration may be topical,parenteral, intravenous, intra-arterial, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracistemal, intraperitoneal, intranasal, aerosol, bysuppositories, or oral administration.

[0105] Therapeutic formulations may be in the form of liquid solutionsor suspensions; for oral administration, formulations may be in the formof tablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

[0106] Methods well known in the art for making formulations are found,for example, in “Remington: The Science and Practice of Pharmacy” (20thed., ed. A. R. Gennaro AR., 2000, Lippincott Williams & Wilkins).Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel. Theconcentration of the broad spectrum inhibitor in the formulation willvary depending upon a number of factors, including the dosage of thedrug to be administered, and the route of administration.

[0107] The broad spectrum inhibitor may be optionally administered as apharmaceutically acceptable salt, such as a non-toxic acid additionsalts or metal complexes that are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andcalcium, among others.

[0108] Administration of compounds in controlled release formulations isuseful where the broad spectrum inhibitor has (i) a narrow therapeuticindex (e.g., the difference between the plasma concentration leading toharmful side effects or toxic reactions and the plasma concentrationleading to a therapeutic effect is small; generally, the therapeuticindex, TI, is defined as the ratio of median lethal dose (LD₅₀) tomedian effective dose (ED₅₀)); (ii) a narrow absorption window in thegastro-intestinal tract; or (iii) a short biological half-life, so thatfrequent dosing during a day is required in order to sustain the plasmalevel at a therapeutic level.

[0109] Many strategies can be pursued to obtain controlled release inwhich the rate of release outweighs the rate of metabolism of the broadspectrum inhibitor. For example, controlled release can be obtained bythe appropriate selection of formulation parameters and ingredients,including, e.g., appropriate controlled release compositions andcoatings. Examples include single or multiple unit tablet or capsulecompositions, oil solutions, suspensions, emulsions, microcapsules,microspheres, nanoparticles, patches, and liposomes.

[0110] Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

[0111] Formulations for oral use may also be provided as chewabletablets, or as hard gelatin capsules wherein the active ingredient ismixed with an inert solid diluent, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium.

[0112] Pharmaceutical formulations of broad spectrum inhibitor describedherein include isomers such as diastereomers and enantiomers, mixturesof isomers, including racemic mixtures, salts, solvates, and polymorphsthereof.

[0113] The formulations can be administered to human patients intherapeutically effective amounts. For example, when the broad spectruminhibitor is an antimicrobial drug, an amount is administered whichprevents, stabilizes, eliminates, or reduces a microbial infection.Typical dose ranges are from about 0.01 μg/kg to about 2 mg/kg of bodyweight per day. The exemplary dosage of drug to be administered islikely to depend on such variables as the type and extent of thedisorder, the overall health status of the particular patient, theformulation of the compound excipients, and its route of administration.Standard clinical trials maybe used to optimize the dose and dosingfrequency for any particular broad spectrum inhibitor.

[0114] The following examples are meant to illustrate, but in no waylimit, the claimed invention.

EXAMPLE I

[0115] This example illustrates the method by whichexperimentally-determined crystal structures of the same inhibitor incomplex with wild type and mutant species of HIV protease can becompared and analyzed for the existence of a three-dimensionallyconserved substructure.

[0116] The structures of wild type HIV-1 protease and a mutant,V82F/184V, HIV-1 protease, both in complexes with the inhibitor shown inFIG. 1 were determined using conventional x-ray crystallographytechniques. The structures were analyzed by means of (a) an overallsuperposition of the atoms of the protein structures; and, (b) a studyof the distances from atoms of the inhibitors to atoms of the protein.This analysis requires three dimensional atomic coordinates of theprotein structures and of the bound inhibitor.

[0117] The superposition of the protein structures was performed in atwo step process: 1) the distance between all pairs of corresponding Cáatoms (Cá atom of residue number 1 in one protein to Cá atom of residuenumber 1 in the second protein; Cá atom of residue number 2 in oneprotein to Cá atom of residue number 2 in the second protein; and so on)of the polypeptide chains is minimized by means of a least-squarealgorithm; 2) the superposition is refined by minimizing, in aniterative process, the distances between corresponding Cá atoms that arecloser than a given distance (0.25 Å in this example), thus eliminatingregions of the structures having large conformational differences tocompute the superposition parameters. The distances between equivalencedCá atoms after the minimization procedure are shown in Table 4. TABLE 4Distances between equivalent Cá atoms Molecule 1: HIV-1 PR wt: 1Molecule 2: HIV-1 PR V82F/I84V mutant: 1 Molecule 1 Molecule 2 distance[Å] CA PRO 1 CA PRO 1 0.455 CA GLN 2 CA GLN 2 0.434 CA ILE 3 CA ILE 30.418 CA THR 4 CA THR 4 0.317 CA LEU 5 CA LEU 5 0.172 CA TRP 6 CA TRP 60.228 CA GLN 7 CA GLN 7 0.364 CA ARG 8 CA ARG 8 0.166 CA PRO 9 CA PRO 90.057 CA LEU 10 CA LEU 10 0.183 CA VAL 11 CA VAL 11 0.194 CA THR 12 CATHR 12 0.168 CA ILE 13 CA ILE 13 0.146 CA LYS 14 CA LYS 14 0.229 CA ILE15 CA ILE 15 0.266 CA GLY 16 CA GLY 16 0.662 CA GLY 17 CA GLY 17 0.491CA GLN 18 CA GLN 18 0.267 CA LEU 19 CA LEU 19 0.112 CA LYS 20 CA LYS 200.128 CA GLU 21 CA GLU 21 0.190 CA ALA 22 CA ALA 22 0.169 CA LEU 23 CALEU 23 0.218 CA LEU 24 CA LEU 24 0.233 CA ASP 25 CA ASP 25 0.160 CA THR26 CA THR 26 0.200 CA GLY 27 CA GLY 27 0.303 CA ALA 28 CA ALA 28 0.169CA ASP 29 CA ASP 29 0.150 CA ASP 30 CA ASP 30 0.038 CA THR 31 CA THR 310.047 CA VAL 32 CA VAL 32 0.173 CA LEU 33 CA LEU 33 0.194 CA GLU 34 CAGLU 34 0.310 CA GLU 35 CA GLU 35 0.260 CA MET 36 CA MET 36 0.136 CA SER37 CA SER 37 0.494 CA LEU 38 CA LEU 38 0.607 CA PRO 39 CA PRO 39 0.094CA GLY 40 CA GLY 40 0.774 CA ARG 41 CA ARG 41 0.448 CA TRP 42 CA TRP 420.204 CA LYS 43 CA LYS 43 0.596 CA PRO 44 CA PRO 44 0.625 CA LYS 45 CALYS 45 0.541 CA MET 46 CA MET 46 0.643 CA ILE 47 CA ILE 47 0.361 CA GLY48 CA GLY 48 0.240 CA GLY 49 CA GLY 49 0.182 CA ILE 50 CA ILE 50 0.110CA GLY 51 CA GLY 51 0.243 CA GLY 52 CA GLY 52 0.200 CA PHE 53 CA PHE 530.119 CA ILE 54 CA ILE 54 0.255 CA LYS 55 CA LYS 55 0.295 CA VAL 56 CAVAL 56 0.108 CA ARG 57 CA ARG 57 0.129 CA GLN 58 CA GLN 58 0.074 CA TYR59 CA TYR 59 0.372 CA ASP 60 CA ASP 60 0.496 CA GLN 61 CA GLN 61 0.780CA ILE 62 CA ILE 62 0.406 CA LEU 63 CA LEU 63 0.211 CA ILE 64 CA ILE 640.260 CA GLU 65 CA GLU 65 0.193 CA ILE 66 CA ILE 66 0.181 CA CYS 67 CACYS 67 0.518 CA GLY 68 CA GLY 68 0.641 CA HIS 69 CA HIS 69 0.319 CA LYS70 CA LYS 70 0.179 CA ALA 71 CA ALA 71 0.265 CA ILE 72 CA ILE 72 0.350CA GLY 73 CA GLY 73 0.253 CA THR 74 CA THR 74 0.301 CA VAL 75 CA VAL 750.187 CA LEU 76 CA LEU 76 0.186 CA VAL 77 CA VAL 77 0.070 CA GLY 78 CAGLY 78 0.306 CA PRO 79 CA PRO 79 0.047 CA THR 80 CA THR 80 0.470 CA PRO81 CA PRO 81 0.404 CA VAL 82 CA PHE 82 0.556 CA ASN 83 CA ASN 83 0.146CA ILE 84 CA VAL 84 0.196 CA ILE 85 CA ILE 85 0.163 CA GLY 86 CA GLY 860.224 CA ARG 87 CA ARG 87 0.127 CA ASN 88 CA ASN 88 0.048 CA LEU 89 CALEU 89 0.081 CA LEU 90 CA LEU 90 0.197 CA THR 91 CA THR 91 0.226 CA GLN92 CA GLN 92 0.176 CA ILE 93 CA ILE 93 0.151 CA GLY 94 CA GLY 94 0.338CA CYS 95 CA CYS 95 0.233 CA THR 96 CA THR 96 0.305 CA LEU 97 CA LEU 970.089 CA ASN 98 CA ASN 98 0.260 CA PHE 99 CA PHE 99 0.250 CA PRO 101 CAPRO 101 0.227 CA GLN 102 CA GLN 102 0.108 CA ILE 103 CA ILE 103 0.206 CATHR 104 CA THR 104 0.169 CA LEU 105 CA LEU 105 0.125 CA TRP 106 CA TRP106 0.363 CA GLN 107 CA GLN 107 0.296 CA ARG 108 CA ARG 108 0.400 CA PRO109 CA PRO 109 0.173 CA LEU 110 CA LEU 110 0.182 CA VAL 111 CA VAL 1110.085 CA THR 112 CA THR 112 0.123 CA ILE 113 CA ILE 113 0.107 CA LYS 114CA LYS 114 0.368 CA ILE 115 CA ILE 115 0.226 CA GLY 116 CA GLY 116 0.638CA GLY 117 CA GLY 117 0.516 CA GLN 118 CA GLN 118 0.414 CA LEU 119 CALEU 119 0.102 CA LYS 120 CA LYS 120 0.191 CA GLU 121 CA GLU 121 0.206 CAALA 122 CA ALA 122 0.197 CA LEU 123 CA LEU 123 0.231 CA LEU 124 CA LEU124 0.145 CA ASP 125 CA ASP 125 0.235 CA THR 126 CA THR 126 0.311 CA GLY127 CA GLY 127 0.200 CA ALA 128 CA ALA 128 0.102 CA ASP 129 CA ASP 1290.143 CA ASP 130 CA ASP 130 0.261 CA THR 131 CA THR 131 0.172 CA VAL 132CA VAL 132 0.232 CA LEU 133 CA LEU 133 0.103 CA GLU 134 CA GLU 134 0.175CA GLU 135 CA GLU 135 0.190 CA MET 136 CA MET 136 0.220 CA SER 137 CASER 137 0.739 CA LEU 138 CA LEU 138 0.277 CA PRO 139 CA PRO 139 0.325 CAGLY 140 CA GLY 140 0.390 CA ARG 141 CA ARG 141 0.174 CA TRP 142 CA TRP142 0.168 CA LYS 143 CA LYS 143 0.304 CA PRO 144 CA PRO 144 0.194 CA LYS145 CA LYS 145 0.456 CA MET 146 CA MET 146 0.362 CA ILE 147 CA ILE 1470.178 CA GLY 148 CA GLY 148 0.390 CA GLY 149 CA GLY 149 0.434 CA ILE 150CA ILE 150 0.050 CA GLY 151 CA GLY 151 0.199 CA GLY 152 CA GLY 152 0.152CA PHE 153 CA PHE 153 0.455 CA ILE 154 CA ILE 154 0.198 CA LYS 155 CALYS 155 0.470 CA VAL 156 CA VAL 156 0.590 CA ARG 157 CA ARG 157 0.607 CAGLN 158 CA GLN 158 0.465 CA TYR 159 CA TYR 159 0.301 CA ASP 160 CA ASP160 0.294 CA GLN 161 CA GLN 161 0.308 CA ILE 162 CA ILE 162 0.274 CA LEU163 CA LEU 163 0.235 CA ILE 164 CA ILE 164 0.367 CA GLU 165 CA GLU 1650.410 CA ILE 166 CA ILE 166 0.201 CA CYS 167 CA CYS 167 0.409 CA GLY 168CA GLY 168 0.406 CA HIS 169 CA HIS 169 0.410 CA LYS 170 CA LYS 170 0.282CA ALA 171 CA ALA 171 0.273 CA ILE 172 CA ILE 172 0.317 CA GLY 173 CAGLY 173 0.563 CA THR 174 CA THR 174 0.129 CA VAL 175 CA VAL 175 0.237 CALEU 176 CA LEU 176 0.155 CA VAL 177 CA VAL 177 0.240 CA GLY 178 CA GLY178 0.386 CA PRO 179 CA PRO 179 0.340 CA THR 180 CA THR 180 0.335 CA PRO181 CA PRO 181 0.446 CA VAL 182 CA PHE 182 0.343 CA ASN 183 CA ASN 1830.205 CA ILE 184 CA VAL 184 0.262 CA ILE 185 CA ILE 185 0.096 CA GLY 186CA GLY 186 0.118 CA ARG 187 CA ARG 187 0.202 CA ASN 188 CA ASN 188 0.073CA LEU 189 CA LEU 189 0.108 CA LEU 190 CA LEU 190 0.127 CA THR 191 CATHR 191 0.177 CA GLN 192 CA GLN 192 0.175 CA ILE 193 CA ILE 193 0.241 CAGLY 194 CA GLY 194 0.118 CA CYS 195 CA CYS 195 0.375 CA THR 196 CA THR196 0.437 CA LEU 197 CA LEU 197 0.167 CA ASN 198 CA ASN 198 0.178

[0118] Table 4 shows that the 184V, V82F mutations induce structuralchanges relative to the wild type structure in some parts of the enzyme,but that other regions are less affected. The regions of the proteinstructure which are not significantly affected by the amino acidmutations are defined as structurally conserved regions. In the presentexample, the mutations result in localized structural changes in thebackbone of HIV protease over a wide range, from 0.038-0.774 Å.

[0119] The distances between the strongly interacting atoms of theinhibitor to atoms of the wild type and mutant protein, that ishydrogen-bond donors and acceptors, were computed and they are displayedin Table 5. TABLE 5 Distances between atoms of the inhibitor and atomsof the protein HIV PR wt: 1 V82F/I84V: 1 O2-Wat301 2.92 2.89 N1-027 3.363.46 O6-N30 3.30 3.61 06-N29 3.19 3.55 O7-N29 2.84 2.87 O7-OD1 29 3.423.54 O7-O1 3.31 3.19 O3-OD 25 (out) 2.50 2.94 O3-OD 25 (in) 2.65 2.67O3-OD125 (out) 3.27 3.21 O3-OD125 (in) 2.80 2.67 O5-Wat301 2.70 2.79O8-N130 3.16 2.96

[0120] Table 5 shows that the atoms of the inhibitor interact with thesame atoms of the two different proteins, in this case the wild type andV82F/184V mutant HIV proteases. From Table 5, it can be seen that theatoms of the enzymes with which the inhibitor interacts belong to thestructurally conserved regions. The effects of mutations on theprotein-inhibitor interactions can be quantified in terms of thedistances between interacting pairs of atoms from the inhibitor and fromatoms of the three-dimensionally conserved substructure of the protein.These distances are similar in the wild type and in the mutantcomplexes; the average of their differences is only 0.07 Å. The range ofthe differences is 0.02-0.36 Å.

EXAMPLE 2

[0121] This example illustrates the method by whichexperimentally-determined crystal structures of two different inhibitorsin complexes with wild type HIV protease can be compared and analyzedfor the existence of a three-dimensionally conserved substructure. Thestructures of wild type HIV-1 protease in complexes with inhibitor 1 andwith Amprenavir (inhibitor 2) were analyzed by means of (a) an overallsuperposition of the protein structures; and (b) a study of thedistances from atoms of the inhibitors to atoms of the protein.

[0122] The superposition of the protein structures is performed in a twostep process: 1) the distance between all pairs of corresponding Cáatoms (Cá atom of residue number 1 in one protein to Cá atom of residuenumber 1 in the second protein; Cá atom of residue number 2 in oneprotein to Cá atom of residue number 2 in the second protein; and so on)of the polypeptide chains is minimized by means of a least-squarealgorithm; 2) the superposition is refined by minimizing, in aniterative process, the distances between corresponding Cá atoms that arecloser than a given distance (0.25 Å in this example), thus eliminatingregions of the structures having large conformational differences tocompute the superposition parameters. The distances between equivalencedCá atoms after the minimization procedure are shown in Table 6. TABLE 6Distances between equivalent Cá atoms Molecule 1: HIV-1 PR wt: 1Molecule 2: HIV-1 PR wt: 2 Molecule 1 Molecule 2 distance [Å] CA PRO 1CA PRO 1 0.200 CA GLN 2 CA GLN 2 0.320 CA ILE 3 CA ILE 3 0.147 CA THR 4CA THR 4 0.405 CA LEU 5 CA LEU 5 0.225 CA TRP 6 CA TRP 6 0.296 CA GLN 7CA GLN 7 0.317 CA ARG 8 CA ARG 8 0.154 CA PRO 9 CA PRO 9 0.143 CA LEU 10CA LEU 10 0.259 CA VAL 11 CA VAL 11 0.275 CA THR 12 CA THR 12 0.307 CAILE 13 CA ILE 13 0.207 CA LYS 14 CA LYS 14 0.273 CA ILE 15 CA ILE 150.434 CA GLY 16 CA GLY 16 0.469 CA GLY 17 CA GLY 17 0.414 CA GLN 18 CAGLN 18 0.319 CA LEU 19 CA LEU 19 0.161 CA LYS 20 CA LYS 20 0.155 CA GLU21 CA GLU 21 0.196 CA ALA 22 CA ALA 22 0.338 CA LEU 23 CA LEU 23 0.246CA LEU 24 CA LEU 24 0.292 CA ASP 25 CA ASP 25 0.142 CA THR 26 CA THR 260.109 CA GLY 27 CA GLY 27 0.176 CA ALA 28 CA ALA 28 0.193 CA ASP 29 CAASP 29 0.087 CA ASP 30 CA ASP 30 0.118 CA THR 31 CA THR 31 0.111 CA VAL32 CA VAL 32 0.087 CA LEU 33 CA LEU 33 0.306 CA GLU 34 CA GLU 34 0.333CA GLU 35 CA GLU 35 0.399 CA MET 36 CA MET 36 0.296 CA SER 37 CA SER 370.454 CA LEU 38 CA LEU 38 0.451 CA PRO 39 CA PRO 39 0.397 CA GLY 40 CAGLY 40 0.444 CA ARG 41 CA ARG 41 0.535 CA TRP 42 CA TRP 42 0.346 CA LYS43 CA LYS 43 0.442 CA PRO 44 CA PRO 44 0.548 CA LYS 45 CA LYS 45 0.307CA MET 46 CA MET 46 0.320 CA ILE 47 CA ILE 47 0.403 CA GLY 48 CA GLY 480.237 CA GLY 49 CA GLY 49 0.280 CA ILE 50 CA ILE 50 0.206 CA GLY 51 CAGLY 51 0.368 CA GLY 52 CA GLY 52 0.315 CA PHE 53 CA PHE 53 0.378 CA ILE54 CA ILE 54 0.180 CA LYS 55 CA LYS 55 0.149 CA VAL 56 CA VAL 56 0.302CA ARG 57 CA ARG 57 0.098 CA GLN 58 CA GLN 58 0.219 CA TYR 59 CA TYR 590.279 CA ASP 60 CA ASP 60 0.385 CA GLN 61 CA GLN 61 0.431 CA ILE 62 CAILE 62 0.343 CA LEU 63 CA LEU 63 0.473 CA ILE 64 CA ILE 64 0.344 CA GLU65 CA GLU 65 0.456 CA ILE 66 CA ILE 66 0.481 CA CYS 67 CA CYS 67 0.920CA GLY 68 CA CLY 68 0.999 CA HIS 69 CA HIS 69 0.295 CA LYS 70 CA LYS 700.406 CA ALA 71 CA ALA 71 0.446 CA ILE 72 CA ILE 72 0.374 CA GLY 73 CAGLY 73 0.259 CA THR 74 CA THR 74 0.276 CA VAL 75 CA VAL 75 0.165 CA LEU76 CA LEU 76 0.220 CA VAL 77 CA VAL 77 0.202 CA GLY 78 CA GLY 78 0.231CA PRO 79 CA PRO 79 0.131 CA THR 80 CA THR 80 0.374 CA PRO 81 CA PRO 810.472 CA VAL 82 CA VAL 82 0.554 CA ASN 83 CA ASN 83 0.149 CA ILE 84 CAILE 84 0.261 CA ILE 85 CA ILE 85 0.223 CA GLY 86 CA GLY 86 0.130 CA ARG87 CA ARG 87 0.165 CA ASN 88 CA ASN 88 0.103 CA LEU 89 CA LEU 89 0.072CA LEU 90 CA LEU 90 0.076 CA THR 91 CA THR 91 0.114 CA GLN 92 CA GLN 920.115 CA ILE 93 CA ILE 93 0.204 CA GLY 94 CA GLY 94 0.220 CA CYS 95 CACYS 95 0.068 CA THR 96 CA THR 96 0.185 CA LEU 97 CA LEU 97 0.095 CA ASN98 CA ASN 98 0.311 CA PHE 99 CA PHE 99 0.216 CA PRO 101 CA PRO 101 0.455CA GLN 102 CA GLN 102 0.121 CA ILE 103 CA ILE 103 0.120 CA THR 104 CATHR 104 0.109 CA LEU 105 CA LEU 105 0.128 CA TRP 106 CA TRP 106 0.205 CAGLN 107 CA GLN 107 0.229 CA ARG 108 CA ARG 108 0.211 CA PRO 109 CA PRO109 0.195 CA LEU 110 CA LEU 110 0.135 CA VAL 111 CA VAL 111 0.086 CA THR112 CA THR 112 0.166 CA ILE 113 CA ILE 113 0.199 CA LYS 114 CA LYS 1140.333 CA ILE 115 CA ILE 115 0.356 CA GLY 116 CA GLY 116 0.671 CA GLY 117CA GLY 117 0.709 CA GLN 118 CA GLN 118 0.370 CA LEU 119 CA LEU 119 0.258CA LYS 120 CA LYS 120 0.156 CA GLU 121 CA GLU 121 0.250 CA ALA 122 CAALA 122 0.276 CA LEU 123 CA LEU 123 0.103 CA LEU 124 CA LEU 124 0.112 CAASP 125 CA ASP 125 0.078 CA THR 126 CA THR 126 0.057 CA GLY 127 CA GLY127 0.121 CA ALA 128 CA ALA 128 0.098 CA ASP 129 CA ASP 129 0.190 CA ASP130 CA ASP 130 0.302 CA THR 131 CA THR 131 0.073 CA VAL 132 CA VAL 1320.178 CA LEU 133 CA LEU 133 0.147 CA GLU 134 CA GLU 134 0.239 CA GLU 135CA GLU 135 0.101 CA MET 136 CA MET 136 0.235 CA SER 137 CA SER 137 0.391CA LEU 138 CA LEU 138 0.364 CA PRO 139 CA PRO 139 0.532 CA GLY 140 CAGLY 140 0.213 CA ARG 141 CA ARG 141 0.448 CA TRP 142 CA TRP 142 0.133 CALYS 143 CA LYS 143 0.195 CA PRO 144 CA PRO 144 0.082 CA LYS 145 CA LYS145 0.359 CA MET 146 CA MET 146 0.306 CA ILE 147 CA ILE 147 0.076 CA GLY148 CA GLY 148 0.214 CA GLY 149 CA GLY 149 0.205 CA ILE 150 CA ILE 1500.163 CA GLY 151 CA GLY 151 0.287 CA GLY 152 CA GLY 152 0.318 CA PHE 153CA PHE 153 0.125 CA ILE 154 CA ILE 154 0.189 CA LYS 155 CA LYS 155 0.384CA VAL 156 CA VAL 156 0.510 CA ARG 157 CA ARG 157 0.405 CA GLN 158 CAGLN 158 0.139 CA TYR 159 CA TYR 159 0.361 CA ASP 160 CA ASP 160 0.252 CAGLN 161 CA GLN 161 0.414 CA ILE 162 CA ILE 162 0.337 CA LEU 163 CA LEU163 0.202 CA ILE 164 CA ILE 164 0.359 CA GLU 165 CA GLU 165 0.463 CA ILE166 CA ILE 166 0.347 CA CYS 167 CA CYS 167 0.256 CA GLY 168 CA GLY 1680.471 CA HIS 169 CA HIS 169 0.658 CA LYS 170 CA LYS 170 0.489 CA ALA 171CA ALA 171 0.445 CA ILE 172 CA ILE 172 0.396 CA GLY 173 CA GLY 173 0.523CA THR 174 CA THR 174 0.130 CA VAL 175 CA VAL 175 0.156 CA LEU 176 CALEU 176 0.077 CA VAL 177 CA VAL 177 0.129 CA GLY 178 CA GLY 178 0.276 CAPRO 179 CA PRO 179 0.272 CA THR 180 CA THR 180 0.580 CA PRO 181 CA PRO181 0.436 CA VAL 182 CA VAL 182 0.328 CA ASN 183 CA ASN 183 0.180 CA ILE184 CA ILE 184 0.151 CA ILE 185 CA ILE 185 0.104 CA GLY 186 CA GLY 1860.059 CA ARG 187 CA ARG 187 0.058 CA ASN 188 CA ASN 188 0.183 CA LEU 189CA LEU 189 0.164 CA LEU 190 CA LEU 190 0.051 CA THR 191 CA THR 191 0.216CA GLW 192 CA GLN 192 0.162 CA ILE 193 CA ILE 193 0.158 CA GLY 194 CAGLY 194 0.047 CA CYS 195 CA CYS 195 0.050 CA THR 196 CA THR 196 0.200 CALEU 197 CA LEU 197 0.165 CA ASN 198 CA ASN 198 0.074

[0123] The distances between the atoms of the inhibitors 1 and 2 toatoms of the protein, that is, hydrogen-bond donors and acceptors, werecomputed and are shown in Table 7. TABLE 7 Distances between atoms ofinhibitors and atoms of the proteins Wt: 1 complex Wt: 2 complexO2-Wat301 2.92 3.02 N1-027 3.36 3.58 O6-N30 3.30 3.50 06-N29 3.19 3.51O7-N29 2.84 — O7-OD1 29 3.42 — O7-O1 3.31 — O3-OD 25 (out) A 2.50 2.80O3-OD 25 (in) A 2.65 2.66 O3-OD 25 (out) B 3.27 3.07 O3-OD 25 (in) B2.80 2.68 O5-Wat301 2.70 2.77 O8-N 30 3.16 — N3-N 30 3.17 N3-OD2 30 3.15

[0124] Inhibitors 1 (FIG. 1) and 2 (Amprenavir) have similar structuralelements, in particular their core, i.e. groups at the P1-P1′60positions. However, 2 has a THF group while 1 has a bis-THF group at theP2′ position. The P2 groups are identical except for the substitution ofan ether oxygen atom in 1 as compared to an amine nitrogen atom at thesame position in 2. Table 7 shows that 1 forms more interactions withthe atoms of the protein that were previously identified as belonging tothe structurally conserved substructure than does compound 2. Forexample, the O7 oxygen atom in compound 1, that forms an interactionwith N29 nitrogen of the protease, has no counterpart in compound 2.Instead, the O6 oxygen atom of 2 forms longer (and presumably weaker)hydrogen bonds with both N30 (3.50 Å) and N29 (3.51 Å). In contrast, theO6 oxygen of compound 1 forms a shorter (and presumably stronger)hydrogen bond with N29 (3.19 Å). Additionally, as can be seen in Table7, where both compounds 1 and 2 form interactions with atoms in thestructurally conserved substructure of HIV protease, the distancesbetween interacting atoms are consistently shorter for compound 1,indicative of presumably stronger binding interactions.

[0125] Examples 1 and 2 were used to identify a threedimensionally-conserved substructure of HIV protease that is involved inthe binding of HIV protease inhibitors and, in particular, to identifyatoms of these substructural elements that are involved in forminginteractions with atoms of HIV protease inhibitors. This substructure isdefined by the set of atomic coordinates (in orthogonal coordinates)provided in Table 8 and any equivalent set derived by applying arbitraryrotations and translations to the set of atomic coordinates in Table 8.The values of the coordinates (X,Y,Z) of the atoms defining thesubstructure are affected by a standard error σ. Therefore (X,Y,Z)values for each atom are those defined in the intervals (X−σ, X+σ) forcoordinate X, (Y−σ, Y+σ) for coordinate Y, and (Z−σ, Z+σ) for coordinateZ. TABLE 8 Three dimensionally-conserved substructure of HIV proteaseAtom X [Å] Y [Å] Z [Å] σ [Å] Description Substructure of the proteinatoms Oxygen −7.9 13.6 27.4 0.5 Oxygen atom of water moleculecoordinated to main chain amide nitrogen atoms of amino acid Gly 49 andGly 149 O27 −13.8 17.7 30.4 0.5 Main Chain carbonyl oxygen atom of aminoacid Gly 27 N29 −13.4 18.2 34.5 0.5 Main chain amide nitrogen atom ofamino acid Asp 29 N30 −11.9 18.6 36.7 0.5 Main chain amide nitrogen atomof amino acid Asp 30 OD1 25 −11.3 21.2 28.7 0.5 Carboxylate oxygen atomof aminoacid Asp 25 OD2 25 −9.4 20.4 29.3 0.5 Carboxylate oxygen atom ofaminoacid Asp 25 OD1 125 −12.7 20.3 26.4 0.5 Carboxylate oxygen atom ofaminoacid Asp 125 OD2 125 −12.7 20.3 26.4 0.5 Carboxylate oxygen atom ofaminoacid Asp 125 N129 −8.9 20.5 20.7 0.5 Main chain amide nitrogen atomof amino acid Asp 129 N130 −10.1 19.5 18.6 0.5 Main chain amide nitrogenatom of amino acid Asp 130 Substructure of the inhibitor atoms Hydrogen−8.8 17.5 25.7 0.5 Interacting with main chain Bond carbonyl oxygen atomof amino donor acid Gly 27 Atom Hydrogen −8.5 15.3 25.1 0.5 Interactingwith Oxygen atom of Bond water molecule coordinated to acceptor mainchain amide nitrogen Atom atoms of amino acid Gly 49 and Gly 149Hydrogen −10.4 19.1 27.4 0.5 Interacting with carboxylate Bond oxygenatoms of aminoacids donor- Asp 25 and Asp 125 acceptor Atom Hydrogen−8.9 14.0 29.8 0.5 Interacting with Oxygen atom of Bond water moleculecoordinated to acceptor main chain amide nitrogen Atom atoms of aminoacid Gly 49 and Gly 149 Hydrogen −8.6 17.3 20.7 0.5 Main chain amidenitrogen atom Bond of amino acid Asp 30 acceptor Atom Hydrogen −6.9 18.721.4 0.5 Interacting with main chain Bond amide nitrogen atom of aminoacceptor acid Asp 29 Atom O8 −10.7 15.8 35.8 0.5 Interacting with mainchain amide nitrogen atom of amino acid Asp 130

EXAMPLE 3

[0126] The following example demonstrates that a protease inhibitor thatcontains atoms that can make favorable interactions with the atoms ofthe substructure may exhibit broad spectrum activity.

[0127] Compounds 1 and 3 contain a Bis-THF group at the P2 position thatcontains two atoms, in particular, hydrogen bond acceptor oxygen atoms,that can form hydrogen bonds with the two hydrogen atoms attached to thebackbone amide nitrogen atoms on the protein at residues 29 and 30.Compound 2 is similar to 1 except that 2 contains a THF group at P2 withonly a single hydrogen bond acceptor oxygen atom. All three compoundsdiffer in the P2′ substituent. Compounds 1 and 3 both are unaffected bythe two active site mutations, V82F and 184V, and Ki values for wildtype and mutant enzymes are similar for both compounds. In contrast,compound 2, which contains only a single hydrogen bond acceptor atom inthe P2 substitutent, is dramatically affected by the active sitemutations, which demonstrate high level resistance to 2.

[0128] The antiviral activity of compounds 1 and 3 against HIV derivedfrom patient isolates that contain multiple mutations are equivalent totheir activity against wild type HIV strains. In contrast, compound 2 ismuch less effective against the same mutant viruses. None of thepatients from whom virus was isolated had ever been exposed to any ofthe compounds tested herein. Nonetheless, compound 2 exhibited crossresistance to these virus strains that is typically seen with allclinically useful HIV protease inhibitors −4 (Saquinavir), 5(Ritonavir), 6 (Indinavir) and 7 (Nelfinavir). Compounds 2, 4, 5, 6, and7 have very different chemical structures, but nonetheless behave as asingle class with respect to their antiviral behavior against wild typeand multidrug resistant HIV strains. All compounds are dramatically lesspotent against the multidrug resistant strains of HIV.

[0129] In sharp contrast, compounds 1 and 3, which closely resemble eachother as well as compound 2, exhibit broad spectrum activity in thatthey are equally effective against wild type and mutant HIV strains thatexhibit high level multidrug resistance towards compounds 2, 4, 5, 6,and 7. The broad spectrum activity of compound 1 was completelyunexpected and contrasts with the common and typical loss of antiviralpotency experienced with compounds like 2, 4, 5, 6, 7, and indeed mostother HIV protease inhibitors represented as similar or differentstructures that have been reported.

[0130] The development and application of the 3D motif method describedabove successfully revealed the presence of a unique, threedimensionally-conserved substructure of HIV protease that is useful inthe design of broad spectrum inhibitors. Based on this method, compound3 was predicted, on the basis of comparative molecular modeling usingthe coordinates of the complexes of compound 1 with wild type andV82F/184V mutant HIV proteases, to be able to make the same keyinteraction as compound 1 and thereby to exhibit broad spectrumactivity. Based on these data, it is feasible to design proteaseinhibitors that are predicted to have broad spectrum activity, and arepredicted to be useful for the treatment of both wild type (first linetherapy) and drug resistant (salvage therapy) HIV infections.

EXAMPLE 4

[0131] This example illustrates the method by whichexperimentally-determined crystal structures of two different targetproteins, DHQases, from two different bacterial species can be comparedand analyzed for the existence of a three-dimensionally conservedsubstructure even in the absence of readily discernible or statisticallysignificant sequence similarity. DHQases from different bacterialspecies typically exhibit less than 30% sequence identity (FIG. 2). Aschematic map showing the key interactions of the substrate-basedinhibitor, DHQO, with the active site residues for the Type II DHQasefrom M. tuberculosis is provided in FIG. 3.

[0132] The structures of wild type DHQase from M. tuberculosis and ahomologous DHQase from Pseudomonas putidas were determined usingconventional x-ray crystallography techniques. The structures wereanalyzed by means of (a) an overall superposition of the atoms of theprotein structures. This analysis requires three dimensional atomiccoordinates of the protein structures.

[0133] The superposition of the protein structures was performed in atwo step process: 1) the distance between all pairs of corresponding Cáatoms (Cá atom of residue number 1 in one protein to Cá atom of residuenumber 1 in the second protein; Cá atom of residue number 2 in oneprotein to Cá atom of residue number 2 in the second protein; and so on)of the polypeptide chains is minimized by means of a least-squarealgorithm; 2) the superposition is refined by minimizing, in aniterative process, the distances between corresponding Cá atoms that arecloser than a given distance (0.4 Å in this example), thus eliminatingregions of the structures having large conformational differences tocompute the superposition parameters. The distances between equivalencedCá atoms after the minimization procedure are shown in Table 9. TABLE 9Distances between equivalent Cá atoms Molecule 1: DHQase P. putida wt:qxa Molecule 2: DHQase M. tuberculosis wt: gt33 Molecule 1 Molecule 2distance [Å] CA MET 2 CA GLU 2 1.078 CA ALA 3 CA LEU 3 1.504 CA THR 4 CAILE 4 1.800 CA LEU 5 CA VAL 5 1.283 CA LEU 6 CA ASN 6 0.911 CA VAL 7 CAVAL 7 0.715 CA LEU 8 CA ILE 8 0.298 CA HIS 9 CA ASN 9 0.211 CA GLY 10 CAGLY 10 0.591 CA PRO 11 CA PRO 11 0.599 CA ASN 12 CA ASN 12 0.487 CA LEU13 CA LEU 13 0.428 CA ASN 14 CA GLY 14 0.229 CA LEU 15 CA ARG 15 0.685CA LEU 16 CA LEU 16 0.541 CA GLY 17 CA GLY 17 1.693 CA THR 18 CA ARG 182.287 CA ARG 19 CA ARG 19 2.956 CA GLN 20 CA GLN 20 3.475 CA PRO 21 CAPRO 21 3.390 CA GLY 22 CA ALA 22 4.037 CA THR 23 CA VAL 23 3.770 CA TYR24 CA TYR 24 2.521 CA GLY 25 CA GLY 25 1.170 CA SER 26 CA GLY 26 1.642CA THR 27 CA THR 27 1.454 CA THR 28 CA THR 28 1.532 CA LEU 29 CA HIS 291.471 CA GLY 30 CA ASP 30 1.632 CA GLN 31 CA GLU 31 1.966 CA ILE 32 CALEU 32 1.586 CA ASN 33 CA VAL 33 1.875 CA GLN 34 CA ALA 34 2.230 CA ASP35 CA LEU 35 2.343 CA LEU 36 CA ILE 36 1.927 CA GLU 37 CA GLU 37 2.284CA ARG 38 CA ARG 38 2.980 CA ARG 39 CA GLU 39 2.917 CA ALA 40 CA ALA 402.719 CA ARG 41 CA ALA 41 3.367 CA GLU 42 CA GLU 42 3.534 CA ALA 43 CALEU 43 3.281 CA GLY 44 CA GLY 44 3.161 CA HIS 45 CA LEU 45 2.899 CA HIS46 CA LYS 46 1.844 CA LEU 47 CA ALA 47 1.599 CA LEU 48 CA VAL 48 1.201CA HIS 49 CA VAL 49 2.053 CA LEU 50 CA ARG 50 1.045 CA GLN 51 CA GLN 510.266 CA SER 52 CA SER 52 0.300 CA ASN 53 CA ASP 53 0.282 CA ALA 54 CASER 54 0.348 CA GLU 55 CA GLU 55 0.326 CA TYR 56 CA ALA 56 0.238 CA GLU57 CA GLN 57 0.380 CA LEU 58 CA LEU 58 0.455 CA ILE 59 CA LEU 59 0.413CA ASP 60 CA ASP 60 0.984 CA ARG 61 CA TRP 61 1.452 CA ILE 62 CA ILE 621.338 CA HIS 63 CA HIS 63 1.310 CA ALA 64 CA GLN 64 2.327 CA ALA 65 CAALA 65 2.526 CA ARG 66 CA ALA 66 3.063 CA ASP 67 CA ASP 67 3.449 CA GLU68 CA CA GLY 69 CA ALA 68 2.318 CA VAL 70 CA ALA 69 1.691 CA ASP 71 CAGLU 70 0.812 CA PHE 72 CA PRO 71 0.515 CA ILE 73 CA VAL 72 0.561 CA ILE74 CA ILE 73 0.547 CA LEU 75 CA LEU 74 0.380 CA ASN 76 CA ASN 75 0.277CA PRO 77 CA ALA 76 0.369 CA ALA 78 CA GLY 77 0.952 CA ALA 79 CA GLY 780.421 CA PHE 80 CA LEU 79 0.714 CA THR 81 CA THR 80 0.575 CA HIS 82 CAHIS 81 0.142 CA THR 83 CA THR 82 0.222 CA SER 84 CA SER 83 0.741 CA VAL85 CA VAL 84 0.719 CA ALA 86 CA ALA 85 0.415 CA LEU 87 CA LEU 86 0.667CA ARG 88 CA ARG 87 0.660 CA ASP 89 CA ASP 88 0.426 CA ALA 90 CA ALA 890.697 CA LEU 91 CA CYS 90 1.233 CA LEU 92 CA ALA 91 1.319 CA ALA 93 CAGLU 92 2.852 CA VAL 94 CA LEU 93 4.165 CA SER 95 CA SER 94 3.605 CA ILE96 CA ALA 95 3.840 CA PRO 97 CA PRO 96 2.414 CA PHE 98 CA LEU 97 0.314CA ILE 99 CA ILE 98 0.251 CA GLU 100 CA GLU 99 0.095 CA VAL 101 CA VAL100 0.131 CA HIS 102 CA HIS 101 0.318 CA ILE 103 CA ILE 102 0.117 CA SER104 CA SER 103 0.229 CA ASN 105 CA ASN 104 0.203 CA VAL 106 CA VAL 1050.193 CA HIS 107 CA HIS 106 0.499 CA LYS 108 CA ALA 107 0.498 CA ARG 109CA ARG 108 0.292 CA GLU 110 CA GLU 109 0.333 CA PRO 111 CA GLU 110 0.377CA PHE 112 CA PHE 111 0.651 CA ARG 113 CA ARG 112 0.611 CA ARG 114 CAARG 113 0.469 CA HIS 115 CA HIS 114 0.467 CA SER 116 CA SER 115 0.293 CATYR 117 CA TYR 116 0.483 CA PHE 118 CA LEU 117 0.468 CA SER 119 CA SER118 0.367 CA ASP 120 CA PRO 119 0.676 CA VAL 121 CA ILE 120 0.445 CA ALA122 CA ALA 121 0.334 CA VAL 123 CA THR 122 0.405 CA GLY 124 CA GLY 1230.372 CA VAL 125 CA VAL 124 0.375 CA ILE 126 CA ILE 125 0.250 CA CYS 127CA VAL 126 0.328 CA GLY 128 CA GLY 127 0.332 CA LEU 129 CA LEU 128 0.473CA GLY 130 CA GLY 129 0.272 CA ALA 131 CA ILE 130 0.551 CA THR 132 CAGLN 131 0.564 CA GLY 133 CA GLY 132 0.289 CA TYR 134 CA TYR 133 0.276 CAARG 135 CA LEU 134 0.476 CA LEU 136 CA LEU 135 0.556 CA ALA 137 CA ALA136 0.677 CA LEU 138 CA LEU 137 0.703 CA GLU 139 CA ARG 138 0.861 CA SER140 CA TYR 139 0.876 CA ALA 141 CA LEU 140 1.330 CA LEU 142 CA ALA 1411.529 CA GLU 143 CA GLU 142 1.492 CA GLN 144 CA HIS 143 1.738 CA LEU 145CA VAL 144 3.487

[0134] Table 9 shows that the two structures are remarkably similaroverall despite their low level sequence identity. However, thestructures exhibit very large deviations in some regions, and are highlyconserved in others. In particular, this analysis reveals that regionsof the enzyme are minimally affected by the large number of amino acidsequence substitutions. The regions of the protein structure which arenot significantly affected by the amino acid substitutions are definedas structurally conserved regions. In the present example, thesubstitutions result in localized structural changes in the backbone ofDHQase over a wide range, from 0.095-4.165 Å.

[0135] The distances between the strongly interacting atoms of theinhibitor to atoms of the homologous DHQase proteins, that is P. putidawt: qxa and M. tuberculosis wt: gt33 complexes, were computed and theyare displayed in Tables 10 and 11, respectively. TABLE 10 Distancesbetween atoms of the inhibitor and atoms of the protein DHQase P. putidawt: gxa distance [Å] C6-PRO11 (O) 3.31 C6-ASN12 (CB) 3.10 N7-TYR24 (OH)2.45 O12-ASN76 (HD2) 2.96 O13-HIS102 (CB) 3.38 O12-SER104 (N) 3.35C6-ASN12 (CB) 3.10

[0136] TABLE 11 Distances between atoms of the inhibitor and atoms ofthe protein DHQase M. tuberculosis wt: gt33 distance [Å] N14-PRO11 (O)3.35 O15-PRO11 (O) 3.01 O15-LEU 13 (CG) 3.36 O15-ARG 19 (NH1) 3.26O15-ARG 19 (NH2) 3.32 O7-ASN 75 (OD1) 2.50 O13-ASN 75 (ND2) 2.98 N14-GLY77 (CA) 3.01 O9-HIS 81 (NE2) 2.82 O7-HIS 101 (ND1) 3.25 O11-ILE 102 (N)3.33 O13-ILE 102 (N) 2.77 O11-SER 103 (N) 2.96 O11-SER 103 (OG) 2.68O9-ARG 112 (NH2) 3.09 O10-ARG 112 (NH2) 3.02

[0137] The methods of Examples 1-3 were applied to the DHQase data toidentify a three dimensionally-conserved substructure of DHQase that isinvolved in the binding of DHQase inhibitors, in particular, to identifythe relevant target substructure for developing broad spectruminhibitors. This substructure is defined by the set of atomiccoordinates (in orthogonal coordinates) provided in Table 12 and anyequivalent set derived by applying arbitrary rotations and translationsto the set of atomic coordinates in Table 12. The values of thecoordinates (X,Y,Z) of the atoms defining the substructure are affectedby a standard error σ. Therefore (X,Y,Z) values for each atom are thosedefined in the intervals (X−σ, X+σ) for coordinate X, (Y−σ, Y+σ) forcoordinate Y, and (Z−σ, Z+σ) for coordinate Z. TABLE 12 Threedimensionally-conserved substructure of DHQase, M. tuberculosis Atom X[Å] Y [Å] Z [Å] σ [Å] Description Substructure of the protein atoms OD126.265 68.912 21.219 0.5 Side chain carbonyl ASN75 oxygen atom of aminoacid ASN 75 ND2 27.336 66.960 20.951 0.5 Side chain nitrogen ASN 75 atomof amino acid ASN 75 NE2 28.343 76.425 22.111 0.5 Side chain nitrogenHIS 81 atom of amino acid HIS 81 ND1 28.079 70.604 23.662 0.5 Side chainnitrogen HIS 101 atom of amino acid HIS 101 N ILE 31.227 67.167 22.1680.5 Main chain amide 102 nitrogen atom of atom acid ILE 102 N SER 33.75468.315 21.558 0.5 Main chain amide 103 nitrogen atom of amino acid Ser103 OG SER 33.946 71.059 20.735 0.5 Side chain hydroxyl 103 oxygen atomof amino acid SER 103 Substructure of the inhibitor Hydrogen 29.60068.554 20.298 0.5 Interacting with main bond chain nitrogen atom ofacceptor ILE 102 and side chain atom nitrogen atom of ASN 75 Hydrogen28.031 70.739 20.422 0.5 Interacting with side bond chain oxygen atom ofdonor- ASN75 and side chain acceptor nitrogen atom of atom HIS 101Hydrogen 29.664 74.658 20.493 0.5 Interacting side chain bond nitrogenatom of HIS donor- 81 acceptor atom Hydrogen 31.451 69.835 20.531 0.5Interacting with main bond chain nitrogen atom acceptor and side chainoxygen atom atom of SER 103

Other Embodiments

[0138] All publications and patent applications, and patents mentionedin this specification are herein incorporated by reference.

[0139] While the invention has been described in connection withspecific embodiments, it will be understood that it is capable offurther modifications. Therefore, this application is intended to coverany variations, uses, or adaptations of the invention that follow, ingeneral, the principles of the invention, including departures from thepresent disclosure that come within known or customary practice withinthe art.

Other embodiments are within the claims. What we claim is:
 1. A methodfor the structure-based design of a drug that can act as an inhibitor ofat least two different biological entities, said method comprising thesteps of: (a) providing at least one structure of a wild type targetprotein or an inhibitor-wild type target protein complex; (b) providingat least one structure of a variant target protein or aninhibitor-variant target protein complex; (c) comparing at least onestructure from step (a) with at least one structure from step (b) todetermine whether there exists a common three-dimensionally conservedsubstructure comprising the atomic coordinates of the structurallyconserved atoms of the inhibitors and structurally conserved atoms ofthe target proteins; and (d) if a conserved substructure exists, usingsaid atomic coordinates of said conserved substructure to select acompound having atoms matching those of said structurally conservedatoms of the inhibitors, wherein the selection of said compound isperformed using computer modeling.
 2. A method for the structure-baseddrug design of a broad spectrum inhibitor, said method comprising thesteps of: (a) providing at least one structure of a wild type targetprotein or an inhibitor-wild type target protein complex; (b) providingat least one structure of a variant target protein or aninhibitor-variant target protein complex; (c) comparing at least onestructure from step (a) with at least one structure from step (b) todetermine whether there exists a common three-dimensionally conservedsubstructure comprising the atomic coordinates of the structurallyconserved atoms the target proteins or a common three-dimensionallyconserved substructure comprising the atomic coordinates of thestructurally conserved atoms of the inhibitors and structurallyconserved atoms of the target proteins; and (d) if a conservedsubstructure exists, using said atomic coordinates of said conservedsubstructure to select a compound having atoms matching those of saidstructurally conserved atoms of the inhibitors or to design a compoundthat binds to said target protein, wherein the selection of saidcompound is performed using computer modeling.
 3. The method of claim 1,further comprising the steps of: (e) comparing at least one structurefrom step (a) with at least one structure from step (b) to determinewhether there exists a three-dimensionally non-conserved substructurecomprising the atomic coordinates of the structurally non-conservedatoms of the inhibitors and structurally non-conserved atoms of thetarget proteins; and (f) if a non-conserved substructure exists, usingsaid atomic coordinates of said non-conserved substructure to reject acompound having atoms matching those of said structurally non-conservedatoms of the inhibitors, wherein the rejection of said compound isperformed in conjunction with computer modeling.
 4. The method of claim1, wherein at least two structures from step b are used in step c. 5.The method of claim 4, wherein at least four structures from step b areused in step c.
 6. The method of claim 4, wherein said target proteinscomprise at least two variant forms.
 7. The method of claim 6, whereinsaid target proteins comprise at least four variant forms
 8. The methodof claim 1, wherein the inhibitors in said inhibitor-wild type targetprotein complex and said inhibitor-variant target protein complex arethe same.
 9. The method of claim 1, wherein the inhibitors in saidinhibitor-wild type target protein complex and said inhibitor-varianttarget protein complex are different.
 10. The method of claim 1, whereinsaid inhibitors are competitive inhibitors.
 11. The method of claim 1,wherein said inhibitors are noncompetitive inhibitors.
 12. The method ofclaim 1, wherein said inhibitors are reversible inhibitors.
 13. Themethod of claim 1, wherein said inhibitors are irreversible inhibitors.14. The method of claim 1, wherein said variant target protein is ahomologous target protein.
 15. The method of claim 1, wherein saidvariant target protein is a mutant target protein.
 16. The method ofclaim 1, wherein at least one of said structures is a crystal structure.17. The method of claim 1, wherein at least one of said structures is annmr structure.
 18. The method of claim 1, wherein at least one of saidstructures is derived using computational methods.
 19. The method ofclaim 1, wherein said target protein is expressed in a microbe and saidmicrobe is selected from the group consisting of viruses, bacteria,protozoa, or fungi.
 20. The method of claim 1, wherein said targetprotein is expressed in a neoplasm.
 21. The method of claims 19 or 20,wherein said target protein is selected from the group consisting of anenzyme, a receptor, a structural protein, a component of amacromolecular complex, a component of a metabolic pathway, or anassembly of biological molecules.
 22. The method of claim 21, whereinsaid enzyme is selected from the group consisting of reversetranscriptases, proteases, DNA and RNA polymerases, methylases,oxidases, hydratases, esterases, acyl transferases, helicases,topoisomerases, and kinases.
 23. The method of claim 22, wherein saidenzyme is HIV protease.
 24. The method of claim 23, wherein saidinhibitors are selected from the group consisting of indinavir,nelfinavir, ritonavir, saquinavir, amprenavir, lopinavir, and UIC-94003.25. The method of claim 23, wherein said structurally conserved atoms ofthe inhibitor and structurally conserved atoms of the protease have theatomic structural coordinates as provided in Table
 8. 26. The method ofclaim 22, wherein said enzyme is 3-dehydroquinate dehydratase.
 27. Themethod of claim 26, wherein said structurally conserved atoms of the3-dehydroquinate dehydratase have the atomic structural coordinates asprovided in Table
 12. 28. A compound having a chemical structureselected using the method of claim 19, wherein said compound has broadspectrum activity against wild type and variant microbes.
 29. A compoundhaving a chemical structure selected using the method of claim 20,wherein said compound has broad spectrum activity against wild type andvariant neoplasms.
 30. The compound of claims 28 or 29, wherein saidcompound has an IC_(50, variant)/IC_(50, wild type) ratio of less than20.
 31. The compound of claim 30, wherein saidIC_(50, variant)/IC_(50, wildtype) ratio is less than
 6. 32. Thecompound of claims 28 or 29, wherein said compound has broad spectrumactivity against at least 3 mutant biological entities.
 33. The compoundof claim 28, wherein said compound has broad spectrum activity againstat least 3 different organisms expressing homologous target proteins.34. A pharmaceutical composition comprising a compound of claim 28 and apharmaceutically acceptable carrier or diluent.
 35. A pharmaceuticalcomposition comprising a compound of claim 29 and a pharmaceuticallyacceptable carrier or diluent.
 36. A compound having a chemicalstructure selected using the method of any one of claims 23-25, whereinsaid compound has broad spectrum activity against HIV protease.
 37. Thecompound of claim 36, wherein said compound has anIC_(50, variant)/IC_(50, wild type) ratio of less than
 10. 38. Thecompound of claim 37, wherein said IC_(50, variant)/IC_(50, wild type)ratio is less than
 6. 39. The compound of claim 36, wherein saidcompound has broad spectrum activity against at least 3 mutantbiological entities.
 40. A pharmaceutical composition comprising acompound of claim 36 and a pharmaceutically acceptable carrier ordiluent.
 41. A compound having a chemical structure selected using themethod of claims 26 or 27, wherein said compound has broad spectrumactivity against 3-dehydroquinate dehydratase.
 42. The compound of claim41, wherein said compound has an IC_(50, variant)/IC_(50, wild type)ratio of less than
 20. 43. The compound of claim 42, wherein saidIC_(50, variant)/IC_(50, wild type) ratio is less than
 10. 44. Thecompound of claim 43, wherein said compound has broad spectrum activityagainst at least 3 mutant biological entities.
 45. The compound of claim41, wherein said compound has broad spectrum activity against at least 3different organisms expressing homologous target proteins.
 46. Apharmaceutical composition comprising a compound of claim 41 and apharmaceutically acceptable carrier or diluent.
 47. A method of treatinga microbial infection in a patient, said method comprising the step ofadministering to said patient a pharmaceutical composition of claim 34in an amount effective to prevent or treat said infection.
 48. A methodof treating a neoplasm in a patient in need thereof, said methodcomprising the step of administering to said patient a pharmaceuticalcomposition of claim 35 in amounts effective to treat said neoplasm. 49.A method of treating an HIV infection in a patient in need thereof, saidmethod comprising the step of administering to said patient apharmaceutical composition of claim 40 in amounts effective to treatsaid infection.
 50. A method of treating a bacterial infection in apatient in need thereof, said method comprising the step ofadministering to said patient a pharmaceutical composition of claim 46in amounts effective to treat said infection.
 51. The method of claim50, wherein said bacterial infection is caused by a bacterium selectedfrom the group consisting of C jejuni, V. cholerae, Y pestis, B.anthracis, P. putidas, and M. tuberculosis.